|Ahead of print
|Neuroendocrine-immune regulating mechanisms for the anti-inflammatory and analgesic actions of acupuncture
Zhi-Fang Xu1, Shou-Hai Hong2, Shen-Jun Wang1, Xue Zhao1, Yang-Yang Liu1, Sha-Sha Ding2, Yuan Xu1, Kuo Zhang2, Nan-Nan Yu2, Zhong-Xi Lv2, Fu-Ming Yang2, Yi-Nan Gong2, Qin-Qin He2, Kun Yu2, Yi-Ping Zhang2, Bao-Min Dou2, Lin Yao2, Ya-Wen Yan2, Tao Yang2, Yan-Fang Zhang2, Bao-Hu Liu2, Yong-Ming Guo1, Hsuah-Nee-Connie Wong2, Yi Guo3
1 Research Center of Experimental Acupuncture Science, Tianjin University of Traditional Chinese Medicine; National Clinical Research Center for Chinese Medicine Acupuncture and Moxibustion, Tianjin, China
2 Research Center of Experimental Acupuncture Science, Tianjin University of Traditional Chinese Medicine, Tianjin, China
3 Research Center of Experimental Acupuncture Science, Tianjin University of Traditional Chinese Medicine; National Clinical Research Center for Chinese Medicine Acupuncture and Moxibustion; College of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, China
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|Date of Submission||26-Mar-2020|
|Date of Acceptance||19-May-2020|
|Date of Web Publication||15-Oct-2020|
Pain and inflammatory diseases are important clinical indications of acupuncture, which have been widely accepted in the international community. Previous studies have been focusing on rapid analgesia of acupuncture through the regulation of nervous system, but few studies on the inflammation regulatory mechanisms in which acupuncture inhibits the peripheral sensitization-induced pain. Based on studies concerning acupoint mechanisms of acupuncture actions and related researches on acupuncture regulating neuroendocrine and immune systems, we put forward the scientific hypothesis that acupuncture regulates neuroendocrine-immune (NEI) network and key response media therein, so as to achieve anti-inflammatory and analgesic effects in target organs. We have established a platform for acupuncture at ST36 to alleviate inflammatory pain in adjuvant induced arthritic rats. Based on the complex network analysis of multi-dimensional data from multi-time point and multi-site detection of NEI common signaling molecules, we have clarified the regulatory effects of acupuncture on NEI network and corresponding downstream immune network. Results indicated that monocytes/macrophages are the key targeting cells of acupuncture regulation, and acupuncture may display the anti-inflammatory and analgesic effects by regulating polarization of T cells in lymph nodes and polarization of M1/M2 macrophages in inflamed joints/paws. In addition, we have spotted a key molecule for acupuncture analgesia, CXCL1, as well as clarified the novel central analgesic mechanism of acupuncture mediated by CXCL1/CXCR2 desensitization. Thereby, we have provided novel evidence of the anti-inflammatory and analgesic actions of acupuncture through regulating NEI network and several key substances, highlighting a systemic research paradigm for investigating mechanisms of acupuncture actions.
Keywords: Acupuncture, anti-inflammation, analgesia, neuro-endocrine-immune network, CXCL1
|How to cite this URL:|
Xu ZF, Hong SH, Wang SJ, Zhao X, Liu YY, Ding SS, Xu Y, Zhang K, Yu NN, Lv ZX, Yang FM, Gong YN, He QQ, Yu K, Zhang YP, Dou BM, Yao L, Yan YW, Yang T, Zhang YF, Liu BH, Guo YM, Wong HN, Guo Y. Neuroendocrine-immune regulating mechanisms for the anti-inflammatory and analgesic actions of acupuncture. World J Tradit Chin Med [Epub ahead of print] [cited 2022 Jan 19]. Available from: https://www.wjtcm.net/preprintarticle.asp?id=298246
| Introduction|| |
Pain and inflammatory diseases are important clinical indications of acupuncture, and they have been widely accepted in the international community. The World Health Organization (WHO) has recommended acupuncture for the treatment of 16 inflammatory diseases such as rheumatoid arthritis (RA), allergic rhinitis, acute and chronic gastritis, periarthritis of shoulder, and more than 30 pain conditions including postoperative pain, low back pain, and fibromyalgia. In 2018, a nationwide survey of acupuncture clinics in the United States suggested inflammatory diseases accounting for 41% of patients treated with acupuncture, and more than 80% of acupuncturists have used acupuncture to treat pain. At the same time, the abuse of opioid analgesics has aroused widespread concern, and many authorities, such as the American College of Physicians, have recommended acupuncture and other non-drug therapies as the first choice for the treatment of pain. In addition, clinical practice guidelines for diseases in several countries also recommended acupuncture for the treatment of various inflammatory diseases and pain conditions, such as myositis, arthritis, allergic rhinitis, and low back pain. Therefore, anti-inflammatory and analgesic mechanisms should be important common pathways of acupuncture effects.
Pain is closely related to inflammation: the local release of serotonin, bradykinin, prostaglandin, and other mediators in inflammatory response can stimulate nerve endings, causing peripheral sensitization of pain. In the pathogenesis of chronic pain such as central pathologic pain, the inflammatory response of microglia is one of the critical mechanisms of pain-associated central sensitization. Therefore, inflammatory response is one of the primary causes of pain, and pain is an important manifestation of the inflammatory response, thus inhibiting the inflammatory response can fundamentally relieve pain. However, previous researches on acupuncture analgesia and anti-inflammatory are mostly carried out separately. Most of the analgesia researches focused on the nerve system, particularly the central analgesic mechanisms. Combined with our previous mechanism studies of acupuncture action, the relevant reports of neuroendocrine and anti-inflammatory regulation by acupuncture, we put forward the acupuncture may modulate neuroendocrine-immune (NEI) network and associated key mediators, and transmit the information to regulate the immune network in the target organs, finally achieving anti-inflammatory and analgesic actions. We replicated the rat model of RA involving NEI network imbalance, and established an experimental platform of anti-inflammatory and analgesic effect of acupuncture. Subsequently, we applied bioinformatics analysis using complex networks to systematically investigate the regulatory effect of acupuncture on NEI network in the circulation and immune network in inflamed joints of RA rats, and verified mediating mechanisms of acupuncture's anti-inflammatory and analgesic effects by focusing on the screened key cells and key molecules.
| Acupuncture Displays Anti-Inflammatory and Analgesic Actions in Adjuvant Induced Arthritic Rats|| |
Establishing a stable platform of acupuncture effect is an important premise to explain the mechanism of acupuncture actions. Based on literature research and previous experiments, as well as taking optimal acupuncture parameters, efficacy evaluation index, and suitable model for acupuncture effect into account, we have established an inflammatory pain model (adjuvant induced arthritis [AIA]), which is a classic RA model. RA is an autoimmune inflammatory disease with joint redness, swelling, and heat pain (inflammatory pain) as its main clinical features. In 2002, the WHO recommended RA as one of the indications for acupuncture, and RA is also an internationally recognized research platform for acupuncture analgesia. On the basis of successfully replicating AIA rats as an inflammatory pain model, we established an effective platform applying manual acupuncture at ST36 (Zusanli), with thermal pain threshold of inflamed paws, degree of ankle swelling, and histological score of ankle joints as therapeutic indicators. It was found that AIA rats showed persistent inflammatory characteristics such as redness, swelling, heat, and pain 1 day after the injection of complete Freund's adjuvant (CFA). The analgesic effect of acupuncture appeared in early stage, which was obvious on the 1st day after modeling and lasted 21 days. The anti-inflammatory effect appeared relatively late, with detumescence trend appeared in 1 week of treatment, and the effect was obvious after 2 weeks. With acupuncture treatment continues, the anti-inflammatory and analgesic effects become more and more obvious, showing time-effect pattern, which lays a foundation for the follow-up mechanism study of acupuncture actions. In addition, the pathological results of ankle joints at day 21 demonstrated that acupuncture could alleviate the inflammatory cell infiltration, cartilage and bone destruction of the inflamed ankle joint of AIA rats to a certain extent, and reduced the contents of pro-inflammatory factors such as interleukin (IL)-1, tumor necrosis factor-alpha (TNF-α), and IL-18 in local joints.
From the above results, acupuncture analgesia can be divided into fast and slow effect. On day 1 after modeling, acupuncture showed analgesic effect, while the anti-inflammatory effect was not obvious. It is consistent with the previous studies that p38 MAPK/ATF-2/VR-1 signaling in spinal cord mediated the relief effect of acupuncture in AIA-induced inflammatory pain. The anti-inflammatory and analgesic effects of electroacupuncture was not synchronized, and the analgesic effects appeared on day 3, maintaining for 21 days. However, the anti-inflammatory effect of electroacupuncture, appeared later than the analgesic effect, lasted for 14 days. Acupuncture analgesia may have various mechanisms, among which the rapid analgesia may be related to the regulation of central nervous system (CNS) function. Studies have shown that acupuncture can inhibit the expression of c-fos caused by the peripheral inflammation within spinal dorsal horn and reduce the transmission of injurious information in the spinal cord., It can also reduce the activation of n-methyl-d-aspartic acid receptor (NMDARs) and inhibit pain signal transmission by regulating opioid peptide, 5-hydroxytryptamine, norepinephrine, and glutamate in spinal cord and brain. At the same time, acupuncture can increase the level of opioid peptide receptors μ-, δ-, and κ phenotypes in spinal dorsal horn of acute pain model, and reduce NMDA activity and inhibit pain conduction. In addition, electroacupuncture can activate the serotonin A1 receptor in inflammatory pain rats and reduce the phosphorylation of NMDARs. In the brain, acupuncture leads to the release of endogenous morphine peptides, which relieve pain by activating the opioid receptor on GABAergic neurons to inhibit the release of GABA in the rostral ventromedial medulla.
RA is an autoimmune disease with a slow disease course and progressive progression in which the pathogenesis is insidious. The results of our study have shown that the anti-inflammatory effect of acupuncture appeared 1–2 weeks after modeling, with the alleviation of the inflammatory symptoms of joints/paws, and the contents of various pro-inflammatory factors in the ankle decreased on day 15 and 21 after the modeling, indicating that the anti-inflammatory action of acupuncture was slow and lasting. Inflammation plays a key role in the pathogenesis of pain. Cytokines and chemokines released in inflammation lead to increased excitability of sensory neurons and enhance the sensitivity of pain. Therefore, the analgesic effect of acupuncture in the later period is mainly realized through anti-inflammatory action, which is also a kind of etiological treatment by acupuncture. In addition, some studies have shown that acupuncture can inhibit the microglia activation in spinal cord dorsal horn, reduce IL-1 β, IL-6, and TNF-α and NMDARs. Thus we speculate that acupuncture can reduce the pain through inhibiting microglia-induced central sensitization, but not covered by this research, worth further exploration.
| Acupuncture Could Regulate the Organic Neuro-Endocrine-Immune Network and Corresponding Downstream Immune Network, With Monocytes/macrophages as the Key Targeting Cells|| |
Acupuncture regulates the organic neuroendocrine-immune network
From the perspective of system science and complexity science, the exploration of life phenomena and the disease nature has become the frontier and hotspot in the field of life science. The modernization of traditional Chinese medicine (TCM) in recent years shows that it is difficult to explain TCM theory by the specific index of a certain system. The human body itself is a complex system, and the biological basis of acupuncture effect also needs to be understood from the macroscopic, holistic, systematic, and dynamic perspectives. At present, although the study on some disease-specific indicators has been found to mediate the acupuncture actions, the extrapolation and clinical translation of the results are severely limited. The reasons are considered to be related to the results not accord to the overall regulation characterized by acupuncture. The system biology takes all the components and their relations in the biological system as the objects, thus the large-scale dynamic analysis and the design of the biological system are put forward by mathematical method.
In TCM, acupuncture is considered to regulate the overall network of meridians and collaterals to treat diseases. The connection between the acupoint stimulation and therapeutic effect is not linear, but comprises a complex network regulatory system. One of the characteristics of acupuncture is overall regulation, which involves multi-level, multi-link, and multi-target patterns. Therefore, to explain the mechanism of acupuncture by means of reductionism is obviously unable to fully reflect the basic characteristics of acupuncture regulation. In 1977, Dr. Berydovsky first proposed the concept of “NEI network” defining that the three systems do not exist in isolation, but are interrelated and interact with each other. NEI network is the biological basis to maintain the organic homeostasis, when the body is in a pathological state, the NEI network rebuilds and plays a self-regulating role, so that, the internal environment tends to be stable and the organism can heal by itself. If the self-regulating ability is exceeded, disease will occur. Acupuncture treatment is closely related to the regulation of NEI complex network. Therefore, to some extent the study of acupuncture regulating NEI network can define the characteristics of the acupuncture action.
Previous studies have shown that acupuncture has a certain regulatory effect on NEI system, but these studies still have some deficiencies. Most studies only focused on some related indicators of nervous, endocrine, and immune systems, but ignored the connection between these indicators, lacking an overall grasp and systematic study of NEI network. There are common signaling molecules and their receptors in these three systems, mainly including some neuropeptides, neurotransmitters, cytokines, hormones, and other signaling molecules and their receptors., For instance, some immune cells secrete not only cytokines but also neurotransmitters and endocrine hormones. In addition to producing neurotransmitters, certain neurons also secrete hormones. It can be seen that these common signaling molecules and their receptors are the important molecular structure foundation of NEI network, enabling the three systems to interact with each other and interact synergistically [Figure 1]. Therefore, these signaling molecules and their receptors, known as the “common chemical language” of NEI network, play a role of information exchange and transmission between the three systems, so that cells can adjust and cooperate with each other to form the NEI cell communication network, maintaining homeostasis. Hence, investigation of NEI common signaling molecules and their crosstalk by acupuncture is helpful to clarify the mechanisms of acupuncture actions.
|Figure 1: Common signaling molecules in the nerve-endocrine-immune network. Common signaling molecules are the important of the nervous, endocrine, and immune system and the network between them|
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Thirty-two kinds of common signaling molecules in NEI network were reported in related domestic and foreign literatures, including 5-hydroxytryptamine, arginine vasopressin, growth hormone (GH) releasing hormone, stem cell factor, corticotropin-releasing hormone, insulin-like growth factor-1, calcitonin gene-related peptide, neuropeptide Y, adrenocorticotrophic hormone, brain-derived neurotrophic factor, follicle stimulating hormone, GH, luteinizing hormone, prolactin (PRL), thyroid-stimulating hormone
(TSH), corticosterone, melatonin, TSH, T3, T4, α-MSH, β-Endorphin, Neurotensin, Orexin A, Oxytocin, Substance P, interferon-gamma (IFN-γ), IL-1α, IL-1 β, IL-2, IL-6, macrophage colony-stimulating factor (M-CSF), and TNF-α [Table 1]. Based on the acupuncture analgesia and anti-inflammatory effects, the serum content of the above 32 factors was detected on day 1, day 7, day 14, and day 21 after modeling. Results showed that on day 1, compared with model group, glucocorticoids concentration was up-regulated and T4 level was down-regulated in acupuncture-treated rats. On day seven, in the acupuncture group, it was found that the concentration of serum IL-6 had increased, whereas concentration of both PRL and substance P had decreased. On day 15, the serum concentration of GH in acupuncture group was decreased compared with that in the model group. As compared with the model group, the serum concentration of GH in the acupuncture group had increased on day 21.
Based on the differences of NEI signaling molecules between groups in serum at four time points, the effects of CFA immunization and acupuncture to NEI cellular communication network were established. Results showed that at different time points after modeling, AIA rats exerted different effects on NEI network, showing a “promotion-repression-promotion-inhibition” trend, which indirectly indicated the innate self-regulation ability. In the meantime, acupuncture showed different effects on NEI network cells in rats at each time point, posing a “inhibition-promotion-inhibition-promotion” trend, which was in contrary to the model group at the same time point. Therefore, we speculate that the effect of acupuncture may be mediated by a two-way benign modulation of NEI communication network.
Acupuncture can regulate the downstream immune network, and monocytes/macrophages were calculated and analyzed as the key target for acupuncture
RA is a systemic autoimmune inflammatory disease in which a variety of innate and acquired immune cells participate in the development of the disease. Antigen activates B cells to produce antibodies, stimulates the activation of T cells, and induces monocytes/macrophages and fibroblast cells to release a large amount of pro-inflammatory medium. In particular, IL-1 and TNF-α can act directly on the synovial fibroblasts, osteoclast and cartilage cells, inducing secretion of matrix metalloproteinases, and eventually lead to the destruction of cartilage and bones of the target organs. The clinical manifestation are joint swelling, pain, and even irreversible damage and deformity of bone and joint, eventually lead to a loss of function.
To explain the mechanism of acupuncture on inflammation and analgesia, we selected 24 immune factors (IL-1α, IL-1 β, IL-6, IL-7, IL-18, TNF-α, IL-2, IL-12, IFN-γ, IL-4, IL-5, IL-10, IL-13, IL-17, CXCL1, MCP-1, RANTES, MIP-1α, MIP-3α, GM-CSF, G-CSF, M-CSF, EPO, vascular endothelial growth factor) which are closely related to the RA pathophysiological development for screening, including innate and acquired immunity factors, chemokines, and related growth factors in serum and ankle homogenate by multiplex immunoassay on day 1, day 7, day 15, and day 21 after modeling. By referring to relevant literatures and authoritative databases, the secretory cells and target cells of differentially signaling molecules were summarized and the cell-cell communication network graph was constructed based on the directed weighted mathematical model, so as to calculate the overall network strength, and reflect the overall cell-cell communication ability in different states and identify the key response cells.
Based on serum data of these 24 immune factors, it was found that the cell-cell communication intensity of AIA rats was enhanced in three time points compared to those of control rats (43.77 at day 1, 60.64 at day 7, 69.55 at day 15), indicating AIA rats displayed an enhancement of immune state. The key immune cells were monocytes/macrophages, Th1, Th2, and B cells, and the key somatic cells were endothelial cells. Compared with AIA group, acupuncture at day 1 enhanced cell-cell communication network intensity (37.66), which was further enhanced at day 7 (111.14), and reduced at day 15 (8.78), this indicates that acupuncture effect on RA could be achieved by immune network enhancement at an early stage and by promoting immune-network to a normal level at a later stage. The cells involved in acupuncture regulation were the same as those in the model group, except that the intensity of intercellular action has changed. Among them, monocytes/macrophages were the key cells of acupuncture, neutrophils, and endothelial cells were also involved in cell communication.
At the same time, we also explored the regulatory effect of acupuncture on the cell-cell communication network of inflammatory ankle joints. Through the analysis of cell-cell communication network modeling, our unpublished results showed that the AIA rats continued to enhance its capability of immune cell communication. As a kind of minimally invasive operation, acupuncture action presented biphasic characteristics, strengthening the local immune cell communication at the early stage, so as to stimulate the body's own immune regulating function, and inhibiting immune cell communication as well as creating anti-inflammatory effect on day 21 anti-inflammatory. In addition, results showed that the acupuncture action on local immune microenvironment of joints was consistent with the data in rat serum. Meanwhile, it was found that the key cells in AIA rat joints were in line with the characteristics of RA disease. At the four observation time points, monocytes/macrophages were critical biproduct induced by acupuncture, which provide an important basis for the further study of anti-inflammatory and analgesic mechanisms of acupuncture in local lesions.
| Acupuncture Regulates T Cell Polarization in Lymph Nodes and M1/m2 Macrophages Polarization in Inflamed Joints by Releasing Transforming Growth Factor-β to Exert Anti-Inflammatory Effect|| |
Acupuncture displays anti-inflammatory and analgesic effects by regulating macrophage polarization in inflammatory organs
Based on the above data of monocytes/macrophages being the key cells of serum and joints for acupuncture regulation, we analyzed the important role of monocytes/macrophages in the pathogenesis of RA and the potential regulatory pathways of acupuncture on them. Monocytes/macrophages are immune cells with strong plasticity, which can be further differentiated into two subtypes of pro-inflammatory M1 and anti-inflammatory M2 under the influence of local microenvironment and display different transcriptional spectrum and functions. This process is known as macrophage polarization which is critical for local inflammation progression and the critical therapeutic target. Among them, M1-phynotype macrophages showed high expression of membrane marker CD86, IL-6, TNF-α, CCL2, inducible nitric oxide synthase and other pro-inflammatory cytokines, aggravating local pathological damage. M2-type macrophages are highly expressed with CD206, arginidase1 and several anti-inflammatory factors or growth transformation factors, such as IL-10, transforming growth factor (TGF)-β to inhibit local inflammatory response and promote tissue repair., Our further research showed that acupuncture did inhibit M1-marcophage population and related factors on day 15 and day 21, thus decreased the proportion of M1/M2. It is confirmed that acupuncture could shift M1 macrophages polarization to M2 phenotypes, thus it can exhibit anti-inflammatory and analgesic action better.
Acupuncture regulated T-cell polarization in lymph nodes and M1/M2 macrophage polarization in inflammatory organs through secretion of transforming growth factor-β
CD4 helper T cells are an important T-cell subset, and the naive helper T cells can differentiate into Th1, Th2, Th17, Treg, and other subgroups with different effects. Existing studies have shown that Th1/Th2/Treg T-cell subsets may be the upstream regulating the mechanism of M1/M2 polarization, in which Th1/Th17 promotes M1-type polarization and Treg/Th2 promotes M2-type polarization., Flow cytometry was used to analyze the T-cell subsets in the popliteal lymph node. The results showed acupuncture could up-regulate Treg cells and related factor TGF-β, which may inhibit the release of M1 macrophages factor to mediate anti-inflammatory action of acupuncture. Due to insufficient anti-rat antibodies for further mechanism investigation, recently, we set up a platform of acupuncture alleviating inflammation of AIA mice models, and found that acupuncture did inhibit the population of pro-inflammatory T cells in the inflammatory lymph nodes, M1 macrophages in the inflamed joint and a variety of pro-inflammatory factors, among which the TGF-β receptor signaling plays an important role in inhibition of M1-type polarization by acupuncture.
In review of previous studies, neurotransmitters/neuropeptides released by the autonomic nerve regulate the polarization of T cells and macrophages. Several studies have found that acupuncture may stimulate the hypothalamic-pituitary-adrenal axis, sympathetic and parasympathetic pathways to fight inflammation. For instance, it was reported that electroacupuncture can effectively cure sepsis by upregulating catechol dopamine and norepinephrine serum levels through vagal circuit. A number of studies also confirmed that the prevention and treatment of diseases by acupuncture are mostly related to autonomic nerve regulation.,, However, the central mechanisms of how acupuncture works on the vagal nerves and/or sympathetic nerves have not been elucidated. Our previous studies have proven that acupuncture in ST36 did not affect the serum content of glucocorticoid, the terminal product of hypothalamic-pituitary-adrenal gland, and had no effect on the polarization of immune cells in the spleen of AIA rats and mice. Thus, we speculate that the immune cell polarization of popliteal lymph nodes is regulated by acupuncture through spinal cord syntagmatic mechanism of ST36. Moreover, there is no parasympathetic nerve distribution in the extremities, so it is speculated that the sympathetic nerve in the same segment of the ST36 is crucial in the anti-inflammatory mechanism of acupuncture.
Our previous studies has shown that the hematopoiesis of tibial bone marrow could be regulated by pituitary adenylate cyclase-activating polypeptide, which was released from direct projecting sympathetic nerve originated from paraspinal sympathetic trunk. Combined the present study on anti-inflammatory mechanism of acupuncture, we further proposed a scientific hypothesis: Needling at ST36 excites local sensory nerve endings, the acupuncture-induced information is transmitted to dorsal root ganglia and spinal cord dorsal horn, then activating the sympathetic neurons at the syntagmatic lateral column, exciting segmental with/in the sympathetic trunk sympathetic neurons, promoting to project popliteal fossa lymph node by sympathetic nerve release neurotransmitters scientific hypothesis [Figure 2], and the related study is undergoing.
|Figure 2: Scientific hypothesis that acupuncture ST36 regulates the immune cell polarization mediated by sympathetic nerve system. Needling at ST36 excites local sensory nerve endings, the acupuncture-induced information is transmitted to dorsal root ganglia and spinal cord dorsal horn, activate the sympathetic neurons at the syntagmatic lateral column, excites segmental with/in the sympathetic trunk sympathetic neurons, promoting to project into the popliteal fossa lymph node sympathetic nerve release neurotransmitters|
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| The Chemotactic Factor Cxcl1 and Cxcl1/cxcr2 Desensitization Displayed as a New Key Signaling Pathway by Acupuncture Analgesia|| |
The chemotactic factor CXCL1 in the blood was identified to mediate acupuncture analgesia
Based on the results shown in section 2, it was found that at 4 different time points after modeling, the serum CXCL1 content was significantly and specifically up-regulated by acupuncture compared with the model group, and a positive correlation was found in the CXCL1 changing trend and acupuncture analgesic effect. Therefore, in order to further clarify the role of circulating CXCL1 in acupuncture analgesia, we recently adopted a pharmacological method by injecting the neutralizing antibody and recombinant CXCL1 protein of different concentrations into the tail vein of AIA rats. Results showed that the analgesic action of acupuncture reduced in AIA rats which received preneutralizing antibody. On the other hand, direct injection of recombinant CXCL1 protein in the blood of AIA rats could play an acupuncture-like analgesic effect, indicating that CXCL1 in blood mediates acupuncture analgesia (unpublished data).
Circulating CXCL1 aggregated into the spinal cord and participated in acupuncture analgesia through CXCL1/CXCR2 desensitization mechanism
Since CXCL1 is significantly elevated in the serum after acupuncture and this mediates the analgesic effect of acupuncture, what are its targets and possible mechanisms of action? we usedin vivo imaging system to observe fluorescence-labeled CXCL1 recombinant protein in AIA rats dynamically to study the migration of circulating CXCL1 in the blood stream. The results showed that CXCL1 could break through the blood-spinal cord barrier and aggregate themselves at the spinal cord. Further experimental studies showed that acupuncture could increase the serum level of chemokine CXCL1, especially in the spinal cord, increasing GRK6, leading to the desensitization of CXCR2 on neurons, and thereby reducing the content of COX2 and PGE2, which are the pain-causing substances in the spinal cord, so as to achieve the analgesic effect of acupuncture (unpublished data).
Liver was confirmed as the main source of CXCL1 in the blood after acupuncture, which was induced by the release of local injury-related molecules into the blood
After we identified where the elevated CXCL1 in the blood “went” after acupuncture, we would like to know where it “came from” - the source of the elevated CXCL1 in the serum of AIA rats after acupuncture. According to previous reports, CXCL1 is widely expressed in organs and tissues such as heart, liver, spleen, lung, kidney, hypothalamus, pituitary, skin, and skeletal muscle, brain, blood and gingiva, and mainly produced by activated monocytes/macrophages, fibroblasts, endothelial cells, and epithelial cells, pancreatic stellate cells, keratinocytes, glial cells, hepatic sinus endothelial cells, cancer cells, peritoneal mesothelial cells, and so on.,,,,,,,,,,, Our results showed that although the CXCL1 protein content of many tissues increased after acupuncture, both CXCL1 gene and the protein levels were increased only in the liver, which was preliminarily believed to be the source of CXCL1 increase in the blood after acupuncture (unpublished data).
Damage-related molecular patterns have been reported to induce the expression of TNF-a in liver macrophages, which would further activate the NF-κB pathway on liver cells and stimulate the high expression of CXCL1 in liver cells. Acupuncture, as a physical stimulus, is bound to cause local injury at the acupuncture point. We found that after acupuncture in normal rats, local HMGB1 was highly expressed in acupoints. Based on the existing evidence, we speculate that “acupuncture → release of HMGB1 in the acupoint → reach the liver through blood circulation → stimulates the secretion of CXCL1 in the liver → increase of CXCL1 in the blood → finally exert the analgesic effect,” and relevant experimental studies are undergoing. Through the above series of studies, we found for the first time that the chemotactic factor CXCL1 in blood mediates the analgesic effect of acupuncture, and the source and downstream mechanism of CXCL1 in blood after acupuncture was explored [Figure 3].
|Figure 3: Scientific hypothesis that acupuncture analgesia is mediated by the chemokine CXCL1. Acupuncture at ST36 → release of HMGB1 in the acupoint → reach the liver through blood circulation → stimulate the secretion of CXCL1 in the liver → increase of CXCL1 in the blood → finally exert the analgesic effect|
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As we known, the treatment of pain is still a great challenge facing the world today. Pain is mediated by the body's injurious sensory nerves, which is a physiological alarm response to protect itself and reduce tissue damage under injury stimuli. However, in pathologic pain conditions, such as chronic pain, the unpleasant experience persists long after the tissue has healed, seriously affecting the patient's life quality., Chronic pain is usually caused by nerve injury and chronic inflammation. This process is accompanied by the release of neurotransmitters, lipid mediators, complement system fragments, neurotrophic factors, cytokines, and chemokines in the CNS and peripheral nervous system. There is increasing evidence that the interaction between the nervous and immune systems leads to sensitization of peripheral and central neurons, which induces and maintains pathological pain.,,,,,,,,,,, Cytokines/chemokines, as the common language of interaction between the two systems, play an important role.,
Cytokines/chemotactic factors are highly active multifunctional small molecule peptides and glycoproteins secreted by various immune cells and nonimmune cells under physiological and pathological conditions and stimulated by various factors. Chemotactic factors are small chemokines that control peripheral immune and/or glial cell transport and migration. Studies have found that chemotactic factors are important mediators for inducing and maintaining pathological pain, especially inflammatory pain, and this effect is not entirely dependent on their chemotaxis. They can regulate the pain process by causing neuronal sensitization. CXCL1, as a chemotactic factor, plays an important role in the regulation of pain. A large number of studies have found that in a variety of pathologic pain, CXCL1 is highly expressed in the brain, spinal cord, dorsal root ganglion of the spinal cord, and inflammatory areas, while down-regulation of its expression, or the expression of its most important receptor CXCR2, can significantly inhibit pathologic pain.,,, In another view, however, CXCL1-activated leukocyte migration to the inflammatory site does not produce pain, and instead, inflammatory site leukocyte opioid analgesia must rely on the recruitment of opioid neutrophils by the CXCR2 chemokine (CXCL1) black,, CXCL1 can preferentially recruit white blood cells containing opioid peptides to migrate to the inflammatory area and bind to CXCR2 receptor on white blood cells to promote the release of opioid peptides and play an important peripheral analgesic and anti-inflammatory role in the inflammatory area. In a neuropathic pain model induced by peripheral nerve injury, investigators also found that increased CXCL1 in the spinal cord has analgesic effects and may be associated with infiltrating neutrophils. In our current unpublished research, it was found that acupuncture significantly increased the level of CXCL1 in the blood of AIA rats, which was then transmitted to the spinal cord and activated GRK6, leading to the desensitization of CXCR2, decreased the expression of CXCR2 receptor on neurons, as well as the content of downstream pain-causing substances COX2 and PGE2, so as to achieve the analgesic effect of acupuncture. The above research has improved theoretical mechanism for acupuncture analgesia from the perspective of immune-nerve interaction.
| Summary|| |
To sum up, acupuncture as a physical stimulus, can prevent and cure diseases by stimulating the organic internal regulatory system. Aiming at the most widely used indications of acupuncture (pain and inflammatory diseases), we proposed that acupuncture can regulate the immune network of diseases, exert anti-inflammatory, and analgesic effect by regulating the organic NEI network. Based on the therapeutic platform of ST36 MA treatment on AIA rats' inflammatory pain, we have innovatively established AIA model and postacupuncture cell communication network applying complex network model, and has revealed the regulating function of acupuncture on NEI network and downstream immune network. In addition, we have spotted monocytes/macrophages as the key cells during acupuncture treatment using computational acupuncture. Furthermore, the new mechanism of anti-inflammatory and analgesic of acupuncture by regulating T-cell polarization in lymph nodes and M1/M2 macrophage polarization in target organs was systematically explained. Chemokine CXCL1, as a new key signaling molecule of acupuncture analgesia, was discovered, and the new mechanism of CXCL1/CXCR2 desensitization-mediated central analgesia was revealed. The potential mechanism of acupuncture anti-inflammatory analgesia was explained from a new perspective of NEI network regulation, and a new paradigm of acupuncture research was provided has been realized.
Financial support and sponsorship
This work was supported by the National Natural Science Foundation of China (NSFC) No. 81330088, 81873369, 81873368, 81973939, 81973944, 81804182; Natural science foundation of Zhejiang province LY20H270008.
Conflicts of interest
There are no conflicts of interest.
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College of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, No. 10 Poyang Lake Road, Tuanbo New Town, Jinghai District, Tianjin 301617
Source of Support: None, Conflict of Interest: None
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